Many diseases (e.g., cancers, hematopoietic disorders, endocrine disorders, and immune disorders) arise from the abnormal expression or activity of a particular gene or group of genes. Similarly, disease can result through expression of a mutant form of protein, as well as from expression of viral genes that have been integrated into the genome of their host. There are numerous therapeutic benefits of the ability to silence selectively these abnormal or foreign genes.
Several therapeutic agents capable of inhibiting the expression of a target gene have been developed, most notably antisense nucleic acid (see, e.g., Skorski, T. et al., Proc. Natl. Acad. Sci. USA (1994) 91:4504-4508). However, antisense approaches, which use either single-stranded RNA or DNA, act in a 1:1 stoichiometric relationship and thus have low efficacy (Skorski et al., supra). For example, Jansen et al. report that relatively high doses (1.7 mg/kg body weight per day, resulting in long term plasma concentrations above 1 mg/L) of antisense RNA specific for the gene bcl2 were required to attain the intended effect of the antisense compound (i.e., 40% reduction in bcl2 expression) (Jansen, B., et al., The Lancet (2000) 356:1728-1733).
Relatively recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression by virtue of a highly conserved regulatory mechanism known as RNA interference (RNAi). Briefly, the RNA III Dicer enzyme processes dsRNA into small interfering RNA (siRNA) of approximately 22 nucleotides. One strand of the siRNA (the “complementary strand”) then serves as a guide sequence to induce cleavage of messenger RNAs (mRNAs) comprising a nucleotide sequence which is at least partially complementary to the sequence of the complementary strand by an RNA-induced silencing complex (RISC) (Hammond, S. M., et al., Nature (2000) 404:293-296). The complementary strand is not cleaved or otherwise degraded in this process, and RISC comprising the complementary strand c an subsequently effect the cleavage of further mRNAs. In other words, RNAi, unlike antisense, involves a catalytic-type reaction and degrades target mRNA in a non-stoichiometric manner. When administered to a cell or organism, exogenous dsRNA has been shown to direct the sequence-specific degradation of endogenous messenger RNA through RNAi. For example, Kreutzer, R., Limmer, S., International PCT Publication No. WO 00/44895 discloses dsRNAs that are effective agents for inducing RNAi, as well as methods for introducing dsRNA into a cell to inhibit the expression of a target gene. Tuschl et al., International PCT Publication No. WO 02/44321 report efficient cleavage of target RNA in a cell lysate using dsRNA having a 3′-nucleotide overhang, thus showing a correlation between the effectiveness of a dsRNA, its length, and the position and length of overhangs of unpaired nucleotides. WO 02/44321 reports improved efficiency when the 3′-nucleotide overhang is 2 nucleotides in length and when the unpaired nucleotide directly adjacent to the terminal nucleotide pair is a uridine base (i.e., a pyrimidine base).
While RNA interference using dsRNA has been shown to be an effective means for selective gene silencing, RNA is extremely unstable in some bodily fluids, particularly in serum. Thus, RNA, including dsRNA, can be degraded between the time it is administered to a subject and the time it enters a target cell. Even within the cell, RNA undergoes rapid degradation by nucleases. Although a more stable or nuclease resistant dsRNA would offer better bioavailability and hence improved effectiveness, the means known to date for stabilizing dsRNA against degradation are insufficient.
Thus, despite significant recent developments in the field of RNA interference, there remains a need for a more effective dsRNA molecule that can selectively and efficiently silence a target gene. More specifically, a dsRNA molecule having enhanced resistance to chemical and/or enzymatic degradation, and hence improved serum stability and bioavailability, and which can be readily and cost-effectively synthesized would be highly desirable. Compositions comprising such agents would be useful for treating diseases caused by abnormal expression or activity of a gene.